Purpose:

c-Fos/activator protein-1 (AP-1) is an important transcription factor for cytokine production and joint destruction in rheumatoid arthritis (RA) and a potential target for the treatment of RA. We have previously reported a small molecule c-Fos/AP-1 inhibitor T-5224 prevented the development of arthritis and joint destruction in mouse collagen-induced arthritis (CIA). The purpose of this study was to investigate the effects of T-5224 on the osteoclastogenesis and bone resorption.

Methods:

1) in vitro: Macrophage-osteoclast precursor RAW264.7 cells were cultured with receptor activator for nuclear factor kB ligand (RANKL) (50 ng/mL) and/or T-5224 for 6 days. Osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRACP)-staining and pit formation assay. The expressions of cathepsin K, c-Fos, and NFATc1 were determined by Western blot analysis. 2) in vivo: CIA was induced in DBA/1J mice by the immunization with bovine type II collagen twice on day 0 and 21. T-5224 was orally administered once daily. The levels of serum TRACP 5b were measured using ELISA, and the mRNA expressions of RANKL and osteoprotegerin (OPG) in the hind paw were measured by RT-PCR. Bone mineral density (BMD) of the femur was assessed using micro-CT. The region of interest was positioned at a metaphyseal region at a point of 3% of the length of the femur from the growth plate.

Results:

T-5224 suppressed the induction of TRACP-positive multinucleated cells under the RANKL-stimulation in RAW264.7 cells. The expressions of cathepsin K and NFATc1 were also inhibited by T-5224 in RANKL-stimulated cells. Meanwhile, the expression level of c-Fos was no difference with or without T-5224. Furthermore, T-5224 inhibited the formation of bone resorption pits in vitro. In mice with CIA, the marked elevation of serum TRACP 5b, a bone resorption marker, was observed on day 35, and this change was significantly suppressed by the administration of T-5224 from day 21. In addition, T-5224 dose-dependently inhibited the increase of RANKL mRNA and the decrease of OPG mRNA in the arthritic hind paws. The BMD of the femora of mice with CIA was lower than that of normal mice on day 50, and T-5224 inhibited the trabecular bone loss.

Conclusions:

These findings suggest that T-5224 inhibits the osteoclastogenesis and bone resorption in arthritic lesions and is able to correct the imbalance of RANKL-OPG which leads to bone loss. Thus, c-Fos/AP-1 inhibitor appears to be a promising drug for rheumatoid bone destruction.